Effect of temperature on the activity of reconstituted influenza virus RNPs. 293T cells were transfected with plasmids for the expression of PB1, PB2, PA and NP (3PNP) and either pPol-I(-)NSCAT (-CAT) or pPol-I(+)NSCAT (+CAT) or the same without PB1 (-PB1). Cells were incubated at 31°C, 37°C or 39°C as indicated for three days. A. Total cellular RNA was isolated and subjected to primer extension analysis for virus-derived CAT m-, c- and vRNA, as labelled. Products of primer extension analysis were separated by 6% denaturing PAGE and detected by autoradiography. The open arrowhead indicates a truncated product derived from vRNA. B. Radiolabeled products for m- c. and vRNA were quantified by densitometry. The ratios of cRNA:mRNA and vRNA:mRNA were calculated and are shown as the fold change (average ± S.D.) in ratios between 31°C and 39°C for cells seeded with vRNA (-CAT) (n = 3) and cRNA (+CAT) (n = 6). C. Doubling dilutions (up to 1/8) of total cell lysates were analysed by electrophoresis and western blotting to detect PB1, NP and clathrin. Purified virions were also included to provide a size marker for viral proteins (lane 1).